We have developed techniques for the isolation of temperature-sensitive mutants of yeast that are simultaneously defective in secretion and cell-surface growth (sec mutants). We have identified 23 complementation groups that are required for the movement of at least two secretory enzymes and one plasma membrane permease through a series of membrane-bound organelles on a pathway that leads to the cell surface. Work in progress is aimed at determining the order in which the sec gene products execute the secretory pathway, the intermediate steps involved in glycoprotein maturation, and the extent to which the secretory pathway contributes to plasma membrane assembly. Goals for the overall project: 1. Isolate a large number of temperature-sensitive growth and secretion mutants (sec). Perform preliminary genetic analysis to allow assignment of complementation groups. 2. Analyze the sec mutants by transmission electron microscopy. Classify the complementation groups by the type of secretory organelle that accumulates. 3. Use histochemical staining procedures to determine if the secretory enzyme, acid phosphatase, accumulates within the secretory organelles. Construct double sec mutants from parent strains that produce distinct cytologic phenotypes at the nonpermissive temperature (37 degrees C). Evaluate the order of events in the pathway by examining the phenotype of the double mutants. 4. Analyze the structure of the oligosaccharide attached to the forms of invertase that are accumulated in the sec mutants. Compare the assembly pathway determined from this analysis with the pathway determined by cytologic and genetic techniques. 5. Evaluate the role of the secretory pathway in plasma membrane assembly. Determine which macromolecules continue or fail to be incorporated into the plasma membrane during incubation of sec mutants at 37 degrees C.